Brassica genomic assays

ABSTRACT

Methods and compositions for detecting, identifying, and quantifying  Brassica  A genomic DNA are described. The methods are specific to the  Brassica  A genome and do not cross-react with other  Brassica  species, crops or weedy relatives that could contribute to contamination of a canola field.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to plant molecular biology.More specifically, it relates to the detection of Brassica A genomicDNA.

BACKGROUND OF THE DISCLOSURE

Brassica species are used as a source of vegetable oil, animal feeds,vegetables and condiments. Brassica plants that are used for vegetableproduction include cabbage, cauliflower, broccoli, kale, kohlrabi, leafmustard and rutabaga. However, on a world-wide basis, the mosteconomically important use of Brassica species is for the production ofseed-derived, vegetable oils. The predominant Brassica species grown foroil production is B. napus, followed by B. juncea and B. rapa. Seeds ofB. napus, B. juncea and B. rapa are referred to as rapeseed. Brassicaspecies that are grown primarily for oil production are often calledoilseed rape. In North America, canola, a type of oilseed rape that hasbeen selected for low levels of erucic acid and glucosinolates in seeds,is the predominant Brassica plant grown for the production of vegetableoil for human consumption.

Canola includes three oilseed Brassica species (B. napus, B. rapa, B.juncea) and is grown on over 80 million acres worldwide Canola is amember of the Brassica genus which includes a wide variety of plantspecies that are under commercial cultivation.

Transgenic canola is currently being cultivated worldwide as a means tosolve agricultural production problems. With the development oftransgenic canola and other transgenic crops, various countries haveinstituted regulations to identify transgenic material and their derivedproducts. Polymerase chain reaction (PCR) methods have generally beenaccepted as the method of choice for transgene detection because of itsquantitative and qualitative reliability. This method usually requiresamplification and detection of a transgene and a corresponding referencegene, and comparing the quantity of the transgene against the quantityof the reference gene. This system requires a set of two primers and adetection probe specific for the transgene and another set of speciesspecific primers and a probe for an endogenous reference gene.

For the purpose of labeling and traceability, transgene detection assaysare developed to meet the requirements of various countries. TheEuropean Union's Regulation 619/2011 specifies that the results ofdetection methods be expressed in transgenic mass fraction with respectto a taxon-specific reference system. The target for the “taxon”specific real-time PCR assay needs to not only be taxon specific, butalso quantitatively stable in different genetic backgrounds in order toyield stable testing results.

In most crops, the target species for developing a specific PCR assay isunique, such as for Zea mays, Glycine max, and Oryza sativa. Forexample, in maize (Zea mays) fields, there are no other closely-relatedZea species likely to cross-contaminate a Zea mays field and thereforecomplicate quantification of maize transgenes. However, canola is quitedifferent.

The Triangle of U (FIG. 1) depicts the evolution and relationshipbetween B. napus, B. rapa, B. juncea and three other Brassica species(Nagaharu U (1935) Genome analysis in Brassica with special reference tothe experimental formation of B. napus and peculiar mode offertilization. Japan. J. Bot 7: 389-452). Through evolution, the 3 basespecies (B. nigra, B. oleracea and B. rapa) have combined to form threeallotetraploid species (B. carinata, B. napus and B. juncea). The threespecies where canola exists (B. juncea, B. napus and B. rapa) share theA-genome (FIG. 1).

If an endogenous system can be proven to specifically detect theA-genome, it could provide an endogenous reference system for a varietyof applications including the relative quantitation of transgenic canolain B. juncea, B. napus and B. rapa. For example, currently mostcommercial transgenic canola is B. napus, however, an A-specificendogenous reference system could be utilized in detection methods onfuture transgenics in the other two species (B. rapa and B. juncea). Inaddition to being able to detect a wide range of varieties of B. rapa,B. napus and B. juncea, the assay must not detect B. nigra, B. carinata,B. oleracea, and other related species that might contaminate a canolagrain lot or other major crops, where such cross-detection reduces theaccuracy of the assay. Even more, there are other closely-relatedBrassica relatives that could contaminate canola fields, including, butnot limited to, Camelina sativa, Thlaspi arvense, Erucastrum gallicum,Raphanus raphanistrum, Raphanus sativus, and Sinapis arvensis. As suchfor canola, from a labeling and traceability viewpoint, the challenge isto identify a real-time PCR assay that will be specific to the speciesthat constitute the canola crop.

Several endogenous reference systems currently exist for measuring therelative percentage of genetically modified canola using real-time PCR.However, these systems are not reliable endogenous reference systems (Wuet al., (2010) Comparison of Five Endogenouse Reference Genes forSpecific PCR Detection and Quantification of Brassica napus, J. Agric.Food Chem, 58: 2812-2817). They are not specific for the taxon or cropof interest, and they have not been shown to be stable across a globallyrepresentative sample within the taxon or crop.

This disclosure relates to methods of detection and quantification thatare specific to the Brassica A-genome and does not significantlycross-react with other Brassica species, crops or weedy relatives thatcould contribute to contamination of a canola field. In addition thisendogenous target is stable within each of the three A-genome specieswhen tested on samples from multiple varieties from diverse geographicalregions.

SUMMARY

Compositions and methods for detecting, identifying, and quantifyingBrassica A genomic DNA are provided herein.

A first aspect features a method of detecting and quantifying the amountof Brassica A genomic DNA in a sample. The method comprises specificallyamplifying a genomic DNA fragment of the Brassica A genome, wherein theamplified DNA fragment comprises at least one of the nucleotidesequences of a genomic region selected from the group consisting of SEQID NOS: 25, 26, 27, 28, and 35; and, detecting and quantifying theBrassica A genome from the amplified fragment of the Brassica A genome.

In an embodiment, the amplified fragment of Brassica A genome comprisesthe nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or 31selected from the group consisting of nucleotide position from about 50to about nucleotide position 400, 50 to about nucleotide position 100,50 to about nucleotide position 350, 400 to about nucleotide position350, 400 to about nucleotide position 200, and 400 to about nucleotideposition 100.

In an embodiment, the amplified fragment of Brassica A genome comprises(i) the nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or31; or (ii) a nucleic acid fragment of at least one of SEQ ID NOS: 29,30, or 31, wherein the nucleic acid fragment is selected from the groupconsisting of nucleotide position from about 50 to about nucleotideposition 400, 50 to about nucleotide position 100, 50 to aboutnucleotide position 350, 400 to about nucleotide position 350, 400 toabout nucleotide position 200, and 400 to about nucleotide position 100of SEQ ID NOS: 29, 30, or 31.

In an embodiment, the amplification is performed with a primer paircomprising nucleotide sequences selected from the group consisting ofSEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24.

In an embodiment, the genomic DNA comprises a FatA gene.

In other embodiments, the amplification does not substantially amplifyBrassica B and C genomic DNA.

Another aspect features a method of determining the relative amount of aBrassica transgenic event in a sample. The method comprises performing aBrassica A genome specific polymerase chain reaction, wherein theanalysis includes specifically amplifying a genomic DNA fragment of theBrassica A genome, wherein the amplified DNA fragment comprises at leastone of the nucleotide sequences of a genomic region selected from thegroup consisting of SEQ ID NOS: 25, 26, 27, 28, and 35; determining thetotal amount of Brassica A genomic DNA in the sample; performing anevent specific assay for the transgenic event to determine the amount ofthe transgenic event in the sample; and, comparing the amount of thetransgenic event DNA to the total amount of Brassica A genomic DNA inthe sample.

In an embodiment, the amplified fragment of Brassica A genome comprisesthe nucleotide sequence of at least one of SEQ ID NOS: 29, 30, or 31from nucleotide position about 50 to about 400.

In an embodiment, the amplification is performed with a primer paircomprising nucleotide sequences selected from the group consisting ofSEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24.

In other embodiments, the genomic DNA comprises a FatA gene.

In an embodiment, the amplification does not substantially amplifyBrassica B and C genomic DNA.

Another aspect features a method of determining adventitious presence ofa Brassica transgenic event in a sample. The method comprises obtaininga sample suspected of containing a Brassica transgenic event; performinga Brassica A genome specific polymerase chain reaction, with a primer,wherein the primer binds to a genomic region of the Brassica A genome,the genomic region selected from the nucleotide sequence of at least oneof SEQ ID NOS: 29, 30, or 31 from nucleotide position about 50 to about400; determining the total amount of Brassica A genomic DNA in thesample; performing an event specific quantitative assay for thetransgenic event to determine the amount of the transgenic event DNA inthe sample; and, comparing the amount of the transgenic event to thetotal amount of Brassica in the sample.

In an embodiment, the primer is selected from the group consisting ofSEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24.

Another aspect features an amplicon comprising at least one of thenucleotide sequences of a genomic region selected from the groupconsisting of SEQ ID NOS: 25, 26, 27, 28, and 35 wherein the amplicon isnot larger than 500 base pairs.

Another aspect features an oligonucleotide comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS: 17, 18, 19,20, 21, 22, 23, and 24 wherein the oligo is about 15-500 nucleotides.

Another aspect features a detection kit comprising oligonucleotidescomprising a nucleotide sequence selected from the group consisting ofSEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24 wherein the oligo is lessthan about 50 nucleotides and one or more reaction components to performa quantitative reaction.

Another aspect features a method of determining trait purity of aBrassica trait. The method comprises obtaining a sample of a Brassicatrait; and performing the Brassica A genome specific assay byspecifically amplifying a genomic DNA fragment of the Brassica A genome,wherein the amplified DNA fragment comprises at least one of thenucleotide sequences of a genomic region selected from the groupconsisting of SEQ ID NOS: 25, 26, 27, 28, and 35; and, detecting andquantifying the Brassica A genome from the amplified fragment of theBrassica A genome.

In an embodiment, the trait is selected from the group consisting ofRT73, RT200, MON88302, DP-073496, HCN92, T45 (HCN28), 23-18-17, 23-198,OXY-235, MS1, MS3, MS6, MS8, RF1, RF2, RF3, and Topas 19/2.

In an embodiment, the determination comprises performing a quantitativepolymerase chain reaction.

Another aspect features a method of establishing purity of a Brassicaseed lot, the method comprising performing a polymerase chain reactionwherein the oligonucleotide primers or probes are capable ofdiscriminating the Brassica A genome from the Brassica B and C genomes,wherein the oligonucleotide primers and/or probes bind to a targetregion of the Brassica A genome, the target region comprising anucleotide sequence selected from the group consisting of SEQ ID NOS:25, 26, 27, 28, and 35.

In an embodiment, the oligonucleotide primers and/or probes comprise anucleotide sequence selected from the group consisting of SEQ ID NOS:19, 20, 21, 22, 23, and 24.

Another aspect features a method of quantifying the amount of atransgenic element in a Brassica sample. The method comprises performinga polymerase chain reaction wherein the oligonucleotide primers orprobes are capable of discriminating the Brassica A genome from theBrassica B and C genomes, the oligonucleotide primers or probes bind toa target region of the Brassica A genome comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS: 29, 30, and31; performing the transgenic element specific quantitative polymerasechain reaction; and, determining the amount of the transgenic elementpresent in the canola sample by comparing to the amount of Brassica Agenomic DNA in the sample.

A further aspect features a method of determining seed purity of a seedsample suspected of containing the Brassica A genome. The methodcomprises specifically amplifying a genomic DNA of the Brassica Agenome, wherein the amplified DNA comprises at least one of thenucleotide sequences of a genomic region selected from the groupconsisting of SEQ ID NOS: 25, 26, 27, 28, and 35; and, determining thepurity of the seed sample based on the presence or absence of theBrassica A genome.

In other embodiments, the seed sample contains at least one of broccoli,brussel sprouts, mustard seeds, cauliflower, collards, cabbage, kale,kohlrabi, leaf mustard, or rutabaga.

In other embodiments, the seed sample contains, but is not limited to,broccoli, brussel sprouts, mustard seeds, cauliflower, collards,cabbage, kale, kohlrabi, leaf mustard, or rutabaga.

Another aspect features a method of determining the presence and/orquantity of the Brassica A genome. The method comprises specificallyhybridizing DNA of the Brassica A genome with a probe, wherein the probeselectively binds to at least one of the nucleotide sequences of agenomic region selected from the group consisting of SEQ ID NOS: 25, 26,27, 28, and 35, optionally under high stringency conditions; and,detecting the presence and/or quantity of the Brassica A genome.

In an embodiment, the probe binds the genomic region of the Brassica Agenome selected from the nucleotide sequence of at least one of SEQ IDNOS: 29, 30, or 31 from nucleotide position about 50 to about 400.

In a further embodiment, the probe comprises the nucleotide sequences ofSEQ ID NOS: 19, 20, 21, 22, 23, or 24.

Another aspect features a method of sequencing a region of the BrassicaA genome. The method comprises obtaining a DNA sample; and, performing asequencing reaction of the DNA sample wherein the sequenced regioncomprises at least one of the nucleotide sequences of a genomic regionselected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and35.

The methods and compositions disclosed herein discriminate detection ofBrassica A genome as compared to detecting genomic DNA of other genomes(e.g., Brassica B and Brassica C genomes).

BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTING

The disclosure can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

FIG. 1 shows the triangle of “U” which depicts the relationships betweenthe different plant species of Brassica (Nagaharu U (1935) Genomeanalysis in Brassica with special reference to the experimentalformation of B. napus and peculiar mode of fertilization. Japan. J. Bot7: 389-452). Number of chromosomes is represented by n. B. nigra, B.oleracea and B. rapa are three base species. Allotetraploid species areB. carinata, B. juncea, and B. napus.

FIG. 2-A shows the evolutionary relationship of the six consensussequences of FatA.

FIG. 2-B shows the breakdown of the 53 sequenced varieties into the sixconsensus sequences.

FIG. 3 shows an alignment of the FatA consensus sequences. The circlesshow regions or bases of A-specificity. The gray highlighted bases showlocation of the primers and probe for the FatA(A) real-time PCR assay.From left to right: the first, second and third highlighted regionsrepresent the forward primer, the probe, and the reverse primer,respectively. The underline bases show where there are ambiguitieswithin the consensus. The sequences that are aligned in the figure arethe A1 sequence (SEQ ID NO: 29), A3 (SEQ ID NO: 31), A2 (SEQ ID NO: 30),B (SEQ ID NO: 32), C2 (SEQ ID NO: 34), and C1 (SEQ ID NO: 33).

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the FatA-A1 consensus nucleotide sequence from Brassicanapus. Varieties used to create SEQ ID NO: 1 include 45H73, NS1822BC,46A76, NS5536BC, 43A56, NW1717M, NW4219BC, NW4201BC. 436554, 458967,531273, 458941, 469735, 458605, 305278, and 633153.

SEQ ID NO: 2 is the FatA-A1.2 consensus nucleotide sequence fromBrassica rapa. Varieties used to create SEQ ID NO: 2 include Tobin,257229, 163496, 649190, and 390962.

SEQ ID NO:3 is the FatA-A1.3 consensus nucleotide sequence from Brassicajuncea. Varieties used to create SEQ ID NO: 3 include JS0917BC,JS0936BC, JS1056BC, JS1260MC, JS1432MC, 418956, 458942, 603011, and649156.

SEQ ID NO:4 is the FatA-A2.1 consensus nucleotide sequence from Brassicanapus. Varieties used to create SEQ ID NO:4 include 458954, 469735, and311729.

SEQ ID NO:5 is the FatA-A2.2 consensus nucleotide sequence from Brassicarapa. Varieties used to create SEQ ID NO:5 include 163496, 347600,346882, and 390962.

SEQ ID NO:6 is the FatA-A2.3 consensus nucleotide sequence from Brassicarapa. Varieties used to create SEQ ID NO:6 include Reward.

SEQ ID NO:7 is the FatA-A3.1 consensus nucleotide sequence from Brassicarapa. Varieties used to create SEQ ID NO:7 include Tobin, Klondike,Reward, 41P95, 257229, 649159, 163496, and 649190.

SEQ ID NO:8 is the FatA-B.1 consensus nucleotide sequence from Brassicajuncea. Varieties used to create SEQ ID NO:8 include JS0879BC, JS0917BC,JS0936BC, JS1056BC, JS1260MC, JS1432MC, 418956, 458942, 603011, and649156.

SEQ ID NO:9 is the FatA-B.2 consensus nucleotide sequence from Brassicacarinata. Varieties used to create SEQ ID NO:9 include 649155 and597822.

SEQ ID NO:10 is the FatA-B.3 consensus nucleotide sequence from Brassicanigra. Varieties used to create SEQ ID NO:10 include 273638 and 633142.

SEQ ID NO:11 is the FatA-C1.1 consensus nucleotide sequence fromBrassica napus. Varieties used to create SEQ ID NO:11 include 45H73,NS1822BC, 46A76, NS5536BC, 46A56, NW1717M, NW4219BC, NW4201BC, 436554,458967, 458954, 531273, 458941, 469735, 458605, 311729, 305278, and633153.

SEQ ID NO:12 is the FatA-C1.2 consensus nucleotide sequence fromBrassica oleracea. Varieties used to create SEQ ID NO:12 include 28888,29800, 365148, 29790, 28852, and 30862.

SEQ ID NO:13 is the FatA-C1.3 consensus nucleotide sequence fromBrassica oleracea. Varieties used to create SEQ ID NO:13 include 32550.

SEQ ID NO:14 is the FatA-C2.1 consensus nucleotide sequence fromBrassica oleracea. Varieties used to create SEQ ID NO:14 include 249556,29041, 29800, 30862, 30724, and 32550.

SEQ ID NO:15 is the FatA-C2.2 consensus nucleotide sequence fromBrassica carinata. Varieties used to create SEQ ID NO:15 include 649155and 597822.

SEQ ID NO:16 is the FatA-other consensus nucleotide sequence fromBrassica juncea. Variety used to create SEQ ID NO:16 include JS1260MC.

SEQ ID NO:17 is the 09-0-2812 primer used to PCR an approximately 500base product from several varieties from the six Brassica species in theTriangle of “U”. 09-0-2812 corresponds to position 1500-1530 (5′ to 3′)for Genbank FatA sequence for Brassica juncea Accession No. AJ294419.

SEQ ID NO:18 is the 09-0-2813 primer used to PCR an approximately 500base product from several varieties from the six Brassica species in theTriangle of “U”. 09-0-2813 corresponds to position 2226-2197 (5′ to 3′)for Genbank FatA sequence for Brassica juncea Accession No. AJ294419.

SEQ ID NO:19 is the 09-0-3249 assay primer used in the A-specific realtime PCR assay SEQ ID NO:20 is the 09-0-3251 FatA A-genome specific realtime PCR assay primer.

SEQ ID NO:21 is the 09-QP87 probe for the FatA A-genome specific realtime PCR assay.

SEQ ID NO:22 is the 11-0-4046 FatA-A-genome specific gel based PCR assayprimer. 11-0-4046 corresponds to position 1782-1813 (5′ to 3′) forGenbank FatA sequence for Brassica napus Accession No. X87842. 11-0-4046corresponds to position 62-93 (5′ to 3′) for Genbank FatA sequence forBrassica juncea Accession No. AJ294419.

SEQ ID NO:23 is the 11-0-4047 FatA gel-based PCR assay primer. 11-0-4047corresponds to position 2001-1971 (5′ to 3′) for the Genbank FatAsequence for Brassica napus Accession No. X87842). 11-0-4047 correspondsto position 279-252 (5′ to 3′) for GenBank FatA sequence for Brassicajuncea Accession No. AJ294419.

SEQ ID NO:24 is the 11-0-4253 FatA gel-based PCR assay primer. 11-0-4253corresponds to position 1918-1889 (5′ to 3′) for the Genbank FatAsequence for Brassica napua Accession No. X87842. 11-0-4253 correspondsto position 196-167 (5′ to 3′) for the Genbank FatA sequence forBrassica juncea Accession No. AJ294419.

SEQ ID NO:25 is a 14 nucleotide consensus region of SEQ ID NOS: 29, 30,and 31.

SEQ ID NO:26 is a 15 nucleotide consensus region of SEQ ID NOS: 29, 30,and 31.

SEQ ID NO: 27 is a 13 nucleotide consensus region of SEQ ID NOS: 29, 30,and 31.

SEQ ID NO:28 is a 15 nucleotide consensus region of SEQ ID NOS: 29, 30,and 31.

SEQ ID NO:29 is the A1 consensus sequence used for the alignment.

SEQ ID NO:30 is the A2 consensus sequence used for the alignment.

SEQ ID NO:31 is the A3 consensus sequence used for the alignment.

SEQ ID NO:32 is the B consensus sequence used for the alignment.

SEQ ID NO:33 is the C1 consensus sequence used for the alignment.

SEQ ID NO:34 is the C2 consensus sequence used for the alignment.

SEQ ID NO:35 is a 15 nucleotide consensus region of SEQ ID NOS: 29, 30,and 31.

SEQ ID NO: 36 is the CruA 09-O-2809 primer.

SEQ ID NO: 37 is the CruA 09-O-2811 primer.

SEQ ID NO: 38 is the HMG-I/Y 09-0-2807 primer.

SEQ ID NO: 39 is the HMG-I/Y 09-0-2808 primer.

SEQ ID NO: 40 is the CruA MDB510 forward primer.

SEQ ID NO: 41 is the MDB511 reverse primer.

SEQ ID NO: 42 is the CruA TM003 probe.

SEQ ID NO: 43 is the FatA FatA-F forward primer.

SEQ ID NO: 44 is the FatA FatA-R reverse primer.

SEQ ID NO: 45 is the FatA FatA-P probe.

SEQ ID NO: 46 is the HMG-I/Y hmg-F forward primer.

SEQ ID NO: 47 is the HMG-I/Y hmg-R reverse primer.

SEQ ID NO: 48 is the HMG-I/Y hmg-P probe.

SEQ ID NO: 49 is the BnACCg8 acc1 forward primer.

SEQ ID NO: 50 is the BnACCg8 acc2 reverse primer.

SEQ ID NO: 51 is the BnACCg8 accp probe.

SEQ ID NO: 52 is the PEP pep-F forward primer.

SEQ ID NO: 53 is the PEP pep-R reverse primer.

SEQ ID NO: 54 is the PEP pep-P probe.

DETAILED DESCRIPTION

Methods of detection and quantification that are specific to theBrassica A-genome and do not substantially cross-react with otherBrassica species, crops or weedy relatives that could contribute tocontamination of a canola field are disclosed. An endogenous target thatis detected is stable within each of the three A-genome species whentested on samples from multiple varieties from diverse geographicalregions.

Units, prefixes, and symbols are denoted in their International Systemof Units (SI) accepted form. Unless otherwise indicated, nucleic acidsare written left to right in 5′ to 3′ orientation; and amino acidsequences are written left to right in amino to carboxy orientation.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange. Nucleotides may be referred to herein by their one-letter symbolsrecommended by the IUPAC-IUBMB Nomenclature Commission. The termsdefined below are more fully defined by reference to the specificationas a whole. Section headings provided throughout the specification areprovided for convenience and are not limitations to the various objectsand embodiments of the present disclosure.

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety, to the extent they relate to the materialsand methods described herein.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein, the term “comprising” means “including but not limitedto.”

“Plant” includes reference to whole plants, plant organs, plant tissues,plant propagules, seeds and plant cells and progeny of same. Plant cellsinclude, without limitation, cells from seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores.

As used herein, the term “canola” refers to a type of Brassica having alow level of glucosinolates and erucic acid in the seed. Three canolaquality Brassica species exist and include B. napus, B. rapa, B. juncea.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein.

The term “dicot” refers to the subclass of angiosperm plants also knownas “dicotyledoneae” and includes reference to whole plants, plant organs(e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny ofthe same. Plant cell, as used herein includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores.

The term “transgenic plant” refers to a plant that comprises within itsgenome a heterologous polynucleotide. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to refer toany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenic organisms or cells initially so altered, aswell as those created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (i.e.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s), including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. An eventis characterized phenotypically by the expression of the transgene(s).At the genetic level, an event is part of the genetic makeup of a plant.The term “event” also refers to progeny produced by a sexual outcrossbetween the transformant and another variety that include theheterologous DNA. Even after repeated back-crossing to a recurrentparent, the inserted DNA and flanking DNA from the transformed parent ispresent in the progeny of the cross at the same chromosomal location.The term “event” also refers to DNA from the original transformantcomprising the inserted DNA and flanking sequence immediately adjacentto the inserted DNA that would be expected to be transferred to aprogeny that receives inserted DNA including the transgene of interestas the result of a sexual cross of one parental line that includes theinserted DNA (e.g., the original transformant and progeny resulting fromselfing) and a parental line that does not contain the inserted DNA.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can comprise either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500,or 5000 base pair or greater which is located either immediatelyupstream of and contiguous with or immediately downstream of andcontiguous with the original foreign insert DNA molecule.

As used herein, a “probe” is an isolated polynucleotide to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such aprobe is complementary to a strand of a target polynucleotide, in theinstant case, to a strand of isolated DNA from the target sample, from asample that includes DNA e.g., from the trait of interest. Probesinclude not only deoxyribonucleic or ribonucleic acids but alsopolyamides and other probe materials that can specifically detect thepresence of the target DNA sequence.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs refer to their use for amplification of atarget polynucleotide, e.g., by the polymerase chain reaction (PCR) orother conventional nucleic-acid amplification methods. “PCR” or“polymerase chain reaction” is a technique used for the amplification ofspecific DNA segments (see, U.S. Pat. Nos. 4,683,195 and 4,800,159;herein incorporated by reference). Any combination of primers disclosedherein can be used such that the pair allows for the detection ofBrassica A-genome.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide of interest. It is recognized that the hybridizationconditions or reaction conditions can be determined by the operator toachieve this result. This length may be of any length that is ofsufficient length to be useful in a detection method of choice.Generally, 8, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100,200, 300, 400, 500, 600, 700 nucleotides or more, or between about11-20, 20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, or more nucleotides in length are used. Suchprobes and primers can hybridize specifically to a target sequence underhigh stringency hybridization conditions. Probes and primers accordingto embodiments may have complete DNA sequence identity of contiguousnucleotides with the target sequence, although probes differing from thetarget DNA sequence and that retain the ability to specifically detectand/or identify a target DNA sequence may be designed by conventionalmethods. Accordingly, probes and primers can share about 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identityor complementarity to the target polynucleotide, or can differ from thetarget sequence by 1, 2, 3, 4, 5, 6 or more nucleotides. Probes can beused as primers, but are generally designed to bind to the target DNA orRNA and are not used in an amplification process.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” or can itselfbe detected for identifying the polynucleotide of interest in biologicalsamples. Alternatively, a probe can be used during the PCR reaction toallow for the detection of the amplification event (i.e., a Taqman probeor a MGB probe) (so called real time PCR). When the probe is hybridizedwith the polynucleotides of a biological sample under conditions whichallow for the binding of the probe to the sample, this binding can bedetected and thus allow for an indication of the presence of an event inthe biological sample. Such identification of a bound probe has beendescribed in the art. In an embodiment, the specific probe is a sequencewhich, under optimized conditions, hybridizes specifically to a regionwithin the 5′ or 3′ flanking region of the desired location and also maycomprise a part of the foreign DNA contiguous therewith. The specificprobe may comprise a sequence of at least 80%, between 80 and 85%,between 85 and 90%, between 90 and 95%, and between 95 and 100%identical (or complementary) to a specific region of the target DNA.

As used herein, “amplified DNA” or “amplified fragment” or “amplicon”refers to the product of polynucleotide amplification of a targetpolynucleotide that is part of a nucleic acid template.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

The term “single nucleotide polymorphism” or “SNP” is a DNA variationoccurring when a single nucleotide—A, T, C, or G—in the genome (or othershared sequence) differs between members of a species (or between pairedchromosomes in an individual). For example, two sequenced DNA fragmentsfrom different individuals, AAGCCTA to AAGCTTA, contain a difference ina single nucleotide. In this case we say that there are two alleles: Cand T. Almost all common SNPs have only two alleles.

Alleles may be detected using various techniques (Nakitandwe et al.,2007; herein incorporated by reference).

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

Probes and primers hybridize specifically to a target sequence understringency hybridization conditions. Hybridization references include,but are not limited to, Herzer and Englert, 2002 Palmisano et al., 2005;herein incorporated by reference.

The term “under stringent conditions” means that two sequences hybridizeunder moderately or highly stringent conditions. More specifically,moderately stringent conditions can be readily determined by thosehaving ordinary skill in the art, e.g., depending on the length of DNA.The basic conditions are set forth by Sambrook et al., MolecularCloning: A Laboratory Manual, third edition, chapters 6 and 7, ColdSpring Harbor Laboratory Press, 2001 and include the use of a prewashingsolution for nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0), hybridization conditions of about 50% formamide, 2×SSC to 6×SSC atabout 40-50° C. (or other similar hybridization solutions, such asStark's solution, in about 50% formamide at about 42° C.) and washingconditions of, for example, about 40-60° C., 0.5-6×SSC, 0.1% SDS.Preferably, moderately stringent conditions include hybridization (andwashing) at about 50° C. and 6×SSC. Highly stringent conditions can alsobe readily determined by those skilled in the art, e.g., depending onthe length of DNA.

Generally, such conditions include hybridization and/or washing athigher temperature and/or lower salt concentration (such ashybridization at about 65° C., 6×SSC to 0.2×SSC, preferably 6×SSC, morepreferably 2×SSC, most preferably 0.2×SSC), compared to the moderatelystringent conditions. For example, highly stringent conditions mayinclude hybridization as defined above, and washing at approximately65-68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl, 10 mMNaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is0.15 M NaCl and 15 mM sodium citrate) in the hybridization and washingbuffers; washing is performed for 15 minutes after hybridization iscompleted.

It is also possible to use a commercially available hybridization kitwhich uses no radioactive substance as a probe. Specific examplesinclude hybridization with an ECL direct labeling & detection system(Amersham). Stringent conditions include, for example, hybridization at42° C. for 4 hours using the hybridization buffer included in the kit,which is supplemented with 5% (w/v) Blocking reagent and 0.5 M NaCl, andwashing twice in 0.4% SDS, 0.5×SSC at 55° C. for 20 minutes and once in2×SSC at room temperature for 5 minutes. As used herein, “amplified DNA”or “amplicon” refers to the product of nucleic acid amplification of atarget nucleic acid sequence that is part of a nucleic acid template.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell. Genomic regionsrefer to a portion of the genome that is targeted to be specificallydetected for example, through an amplification reaction or by directsequencing.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

The term “homologous” refers to nucleic acid sequences that are derivedfrom a common ancestral gene through natural or artificial processes(e.g., are members of the same gene family), and thus, typically, sharesequence similarity. Typically, homologous nucleic acids have sufficientsequence identity that one of the sequences or its complement is able toselectively hybridize to the other under selective hybridizationconditions. The term “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences have about at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity with each other. A nucleic acid that exhibits at least somedegree of homology to a reference nucleic acid can be unique oridentical to the reference nucleic acid or its complementary sequence.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from amRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

“Regulatory sequences” refer to nucleotide sequences located upstream(5′ non-coding sequences), within, or downstream (3′ non-codingsequences) of a coding sequence, and which influence the transcription,RNA processing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences. The terms “regulatory sequence” and “regulatoryelement” are used interchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably, and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

Amplification

In vitro amplification techniques are well known in the art. Examples oftechniques sufficient to direct persons of skill through such in vitromethods, including the polymerase chain reaction (PCR), the ligase chainreaction (LCR), Qβ-replicase amplification and other RNA polymerasemediated techniques (e.g., NASBA), are found in Berger, Sambrook andAusubel (all supra) as well as Mullis et al. ((1987) U.S. Pat. No.4,683,202); PCR Protocols, A Guide to Methods and Applications ((Inniset al., eds.) Academic Press Inc., San Diego Academic Press Inc. SanDiego, Calif. (1990) (Innis)); Arnheim & Levinson ((Oct. 1, 1990) C&EN36-47); The Journal Of NIH Research (1991) 3, 81-94; Kwoh et al. ((1989)Proc. Natl. Acad. Sci. USA 86, 1173); Guatelli et al. ((1990) Proc.Natl. Acad. Sci. USA 87, 1874); Lomeli et al. ((1989) J. Clin. Chem. 35,1826); Landegren et al. ((1988) Science 241, 1077-1080); Van Brunt((1990) Biotechnology 8, 291-294); Wu and Wallace ((1989) Gene 4, 560);Barringer et al. ((1990) Gene 89, 117), and Sooknanan and Malek ((1995)Biotechnology 13: 563-564). Improved methods of cloning in vitroamplified nucleic acids are described in Wallace et al., U.S. Pat. No.5,426,039. Improved methods of amplifying large nucleic acids by PCR aresummarized in Cheng et al. (1994) Nature 369: 684, and the referencestherein, in which PCR amplicons of up to 40 kb are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. See,Ausubel, Sambrook and Berger, all supra.

In an embodiment of the disclosure described herein, the amplifiedfragment of Brassica A genome comprises (i) the nucleotide sequence ofat least one of SEQ ID NOS: 29, 30, or 31; or (ii) a nucleic acidfragment of at least one of SEQ ID NOS: 29, 30, or 31, wherein thenucleic acid fragment is selected from the group consisting ofnucleotide position from about 50 to about nucleotide position 400, 50to about nucleotide position 100, 50 to about nucleotide position 350,400 to about nucleotide position 350, 400 to about nucleotide position200, and 400 to about nucleotide position 100 of SEQ ID NOS: 29, 30, or31. The amplified fragment of Brassica A genome comprises a nucleic acidfragment of at least one of SEQ ID NOS: 29, 30, or 31, wherein thenucleic acid fragment is selected from the group consisting ofnucleotide position from about 50 to about nucleotide position 400. Thismay include, but is not limited to, any single numeric digit intervalfrom about position 50 to about position 400. The nucleic acid fragmentmay comprise any amplified fragment between and including nucleotideposition 50 to nucleotide position 400.

In an embodiment of the disclosure, the detection method comprisessequencing a biological sample containing genomic DNA of Brassica Agenome, wherein the genomic DNA comprises (i) the nucleotide sequence ofat least one of SEQ ID NOS: 29, 30, or 31; or (ii) a nucleic acidfragment of at least one of SEQ ID NOS: 29, 30, or 31, wherein thenucleic acid fragment is selected from the group consisting ofnucleotide position from about 50 to about nucleotide position 400, 50to about nucleotide position 100, 50 to about nucleotide position 350,400 to about nucleotide position 350, 400 to about nucleotide position200, and 400 to about nucleotide position 100 of SEQ ID NOS: 29, 30, or31 or a complement thereof. The portion of the genomic DNA sequenced canbe any region within one of SEQ ID NOS: 29, 30, or 31.

The amplicon produced by these methods may be detected by a plurality ofmethods.

Oligonucleotides for use as primers, e.g., in amplification reactionsand for use as nucleic acid sequence probes, are typically synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers ((1981) Tetrahedron Lett. 22:1859),or can simply be ordered commercially.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art.

“Sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences refers to residues that are the same in bothsequences when aligned for maximum correspondence over a specifiedcomparison window.

“Percentage sequence identity” refers to the value determined bycomparing two optimally aligned sequences over a comparison window. Thepercentage is calculated by determining the number of positions at whichboth sequences have the same nucleotide or amino acid residue,determining the number of matched positions, dividing the number ofmatched positions by the total number of positions in the comparisonwindow, and multiplying the result by 100 to yield the percentage ofsequence identity.

When percentage of sequence identity is used in reference to proteins itis recognized that residue positions that are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ byconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller (1988) Computer Applic. Biol. Sci.4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman ((1981) Adv. Appl.Math. 2:482); by the homology alignment algorithm of Needleman andWunsch ((1970) J. Mol. Biol. 48:443); by the search for similaritymethod of Pearson and Lipman ((1988) Proc. Natl. Acad. Sci. USA85:2444); by computerized implementations of these algorithms,including, but not limited to: CLUSTAL in the PC/Gene program byIntelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG), Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp ((1988) Gene 73:237-244); Higgins and Sharp ((1989)CABIOS 5:151-153); Corpet et al. ((1988) Nucleic Acids Research16:10881-90); Huang et al. ((1992) Computer Applications in theBiosciences 8: 155-65), and Pearson et al. ((1994) Methods in MolecularBiology 24:307-331).

The BLAST family of programs that can be used for database similaritysearches includes: BLASTN for nucleotide query sequences againstnucleotide database sequences; BLASTX for nucleotide query sequencesagainst protein database sequences; BLASTP for protein query sequencesagainst protein database sequences; TBLASTN for protein query sequencesagainst nucleotide database sequences; and TBLASTX for nucleotide querysequences against nucleotide database sequences. See, e.g., CurrentProtocols in Molecular Biology, Chapter 19, Ausubel et al., Eds., (1995)Greene Publishing and Wiley-Interscience, New York; Altschul et al.(1990) J. Mol. Biol. 215:403-410; and, Altschul et al. (1997) NucleicAcids Res. 25:3389-3402.

Software for performing BLAST analyses is publicly available, e.g.,through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, e.g., Henikoff & Henikoff (1989) Proc.Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance.

BLAST searches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequences thatmay be homopolymeric tracts, short-period repeats, or regions enrichedin one or more amino acids. Such low-complexity regions may be alignedbetween unrelated proteins even though other regions of the protein areentirely dissimilar. A number of low-complexity filter programs can beemployed to reduce such low-complexity alignments. For example, the SEG(Wooten and Federhen (1993) Comput. Chem. 17:149-163) and XNU (Clayerieand States (1993) Comput. Chem. 17:191-201) low-complexity filters canbe employed alone or in combination.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

EXAMPLES

The following experimental methods and results provide additionaldetails regarding specific aspects of protocols and procedures relevantto the practice of the present disclosure. The examples, which areprovided without limitation to illustrate the claimed invention, involvethe application of protocols well known to those of skill in the art,and detailed in the references cited herein.

Example 1 Development of A-Genome Specific Endogenous Reference SystemSelection of Plant Material

Seeds from various countries were selected from the six Brassica speciesthat make up the Triangle of “U”: B. carinata, B. juncea, B. napus, B.nigra, B. oleracea, and B. rapa (as well as B. rapa subspecies)varieties (FIG. 1). Brassica-related species that may be found in oraround canola were also included in this study. This includes: Camelinasitava, Erucastrum gallicum, Thlaspi arvense, Sinapis alba and Sinapisarvensis (as well as S. arvensis subspecies). Seeds from other cropswere also included: maize, rice, sorghum, tomato, cotton, soybean. Themajority of seeds were received from Pioneer Hybrid Inc. in Georgetown,Ontario, Canada and also from USDA (United States Department ofAgriculture) in Ames, Iowa and Geneva, N.Y. Table 1 shows the source ofseeds used. For those seeds obtained from the USDA, the USDA AccessionNumbers (ACNO) are included.

TABLE 1 Seed Sources. ACNO (seeds from USDA); Variety name (seeds fromSpecies Source Pioneer) B. napus Pioneer 45H73 Pioneer NS1822BC Pioneer46A76 Pioneer NS5536BC Pioneer 43A56 Pioneer NW1717M Pioneer NW4219BCPioneer NW4201BC USDA (Ames, IA) 436554 USDA (Ames, IA) 458967 USDA(Ames, IA) 458954 USDA (Ames, IA) 531273 USDA (Ames, IA) 458941 USDA(Ames, IA) 469735 USDA (Ames, IA) 458605 USDA (Ames, IA) 311729 USDA(Ames, IA) 305278 B. rapa Pioneer Tobin Pioneer Klondike Pioneer RewardPioneer 41P95 USDA (Ames, IA) 257229 USDA (Ames, IA) 633153 USDA (Ames,IA) 649159 USDA (Ames, IA) 163496 B. rapa ssp USDA (Ames, IA) 347600dichotoma B. rapa ssp oleifera USDA (Ames, IA) 649190 B. rapa ssp USDA(Ames, IA) 346882 trilocularis B. rapa var. USDA (Ames, IA) 390962parachinensis B. juncea Pioneer JS0879BC Pioneer JS0917BC PioneerJS0936BC Pioneer JS1056BC Pioneer JS1260MC Pioneer JS1432MC USDA (Ames,IA) 418956 USDA (Ames, IA) 458942 USDA (Ames, IA) 603011 B. oleraceavar. USDA (Geneva, NY) 249556 alboglabra B. oleracea var. USDA (Geneva,NY) 28888 botrytis B. oleracea var. USDA (Geneva, NY) 29041 capitata B.oleracea var. USDA (Geneva, NY) 29800 costata B. oleracea var. USDA(Geneva, NY) 365148 gemmifera B. oleracea var. USDA (Geneva, NY) 29790gongylodes B. oleracea var. USDA (Geneva, NY) 28852 italica B. oleraceavar. USDA (Geneva, NY) 30862 medullosa B. oleracea var. USDA (Geneva,NY) 30724 ramosa B. oleracea var. USDA (Geneva, NY) 32550 viridis B.nigra USDA (Ames, IA) 273638 USDA (Ames, IA) 633142 USDA (Ames, IA)649156 USDA (Ames, IA) 193960 USDA (Ames, IA) 271444 USDA (Ames, IA)633143 USDA (Ames, IA) 220282 USDA (Ames, IA) 649154 USDA (Ames, IA)280638 USDA (Ames, IA) 357369 USDA (Ames, IA) 633147 USDA (Ames, IA)131512 USDA (Ames, IA) 597829 USDA (Ames, IA) 649155 B. carinata USDA(Ames, IA) 613124 USDA (Ames, IA) 597822 USDA (Ames, IA) 360879 USDA(Ames, IA) 596534 USDA (Ames, IA) 2779 USDA (Ames, IA) 193460 USDA(Ames, IA) 633076 USDA (Ames, IA) 390133 USDA (Ames, IA) 209023 USDA(Ames, IA) 360882 Sinapis alba USDA (Ames, IA) 19266 USDA (Ames, IA)305276 USDA (Ames, IA) 311724 USDA (Ames, IA) 458960 USDA (Ames, IA)409025 USDA (Ames, IA) 633274 Sinapis arvensis USDA (Ames, IA) 21449USDA (Ames, IA) 597863 USDA (Ames, IA) 633374 Sinapis arvensis USDA(Ames, IA) 296079 subsp. arvensis USDA (Ames, IA) 407561 USDA (Ames, IA)633411 Erucastrum USDA (Ames, IA) 22990 gallicum Raphanus USDA (Geneva,NY) 271456 raphanistrum Raphanus sativus USDA (Geneva, NY) 268370Camelina sativa USDA (Ames, IA) 650165 Thlaspi arvense USDA (Ames, IA)633414 USDA (Ames, IA) 29118 Arabidopsis Pioneer thaliana Columbia typeGossypium Pioneer hirsutum Helianthus annuus USDA (Ames, IA) 7451 USDA(Ames, IA) 29348 USDA (Ames, IA) 592319 Solanum USDA (Geneva, NY) 645248lycopersicum Glycine max Pioneer Oryza sativa Grocery store (USA)Sorghum bicolor Pioneer Zea mays Pioneer Italicized text denotes seedvarieties used for sequencing. ACNO represents accession number forseeds acquired from USDA. ACNO = Accession Number; Note: Sequences fromACNO 633153 were assigned to both the A and C contigs, suggesting thisvariety may actually be a B. napus (AACC) variety (see Example 3herein). Two B. nigra varieties (ACNO 633142 and ACNO 649156) wereassigned to two different consensuses suggesting that these twovarieties are not the same species (see Example 3 herein).Genomic DNA Extraction from Seeds

For consistency, all genomic DNA was extracted from seeds using the sameDNA extraction method: a CTAB-based lysis method with passage of theprecipitated DNA through a Qiagen Genomic Tip (Qiagen Inc, Valencia,Calif.) for further purification. This DNA extraction protocol wasvalidated in-house for Zea mays, Gycine max, and Brassica napus. All DNAsamples were quantified using a PicoGreen assay (Molecular Probes;Eugene, Oreg.). For the Specificity Comparison of assays, most of theDNAs were tested in a total of 9 reactions (i.e. 3 runs in triplicate).The exceptions are listed in Table 2.

TABLE 2 Exceptions (DNA tested in less than 9 replicates) ACNO orVariety HMG- name Species BnACCg8 FatA FatA(A) I/Y PEP Arabidopsis 6 4 74 4 thaliana Columbia type JS0879BC Brassica 6 juncea JS0917BC Brassica6 juncea NW4219BC Brassica 6 469735 napus 6 193960 Brassica 4 2 2 2 2220282 nigra 2 2 5 2 2 357369 2 7 7 7 4 597829 6  22990 Erucastrum 6 4 74 4 gallicum 633374 Sinapis 6 6 6 6 6 arvensis 633414 Thlaspi 6 arvense

Primers and Probes

The primers used herein were synthesized by Integrated DNA Technologies(Coralville, Iowa), and the probes were synthesized by AppliedBiosystems (Carlsbad, Calif.). The sequences of all primers used forgenerating PCR products for sequencing are listed in Table 3; and thesequences for all primers and probes for the Specificity Testing arelisted in Table 4. All probes were labeled as described in the relevantreferences, except the HMG-I/Y probe 9. Since the SDS2.3 software doesnot have a detector for HEX, the HMG-I/Y probe was labeled with VICinstead of HEX, since the VIC dye fluoresces in wavelength similar tothe HEX dye.

TABLE 3Primers Used for Generating PCR Products. B. carinata or B. nigra sequenceswere not found for CruA, FatA, or HMG-I/Y genes in Genbank.Genbank Accession numbers B. napus (position of  forward (f) and Primer/ Sequence reverse ® primers;  B. Gene Probe (5′ to 3′)amplicon size) B. rapa B. juncea  oleracea CruA 09-O-2809 AGCTCAATGCACX1455 (f: 809-834; KBrHO42K14F n/a BOGK18 TGGAGCCGTCAC r: 1555-1528; 6TF AC 747 bp) (SEQ ID NO: 36) 09-O-2811 GGTGGCTGGCTA AATCGAGGACG GAAAC(SEQ ID NO: 37) FatA 09-O-2812 GACACAAGGCG X87842 (f: 1721- n/aBJU278479, n/a GCTTCAAAGAGT 1751; r: 2226-2197; AJ294419 TACAGATG506 bp) (SEQ ID NO: 17) 09-O-2813 ACAATGTCATCT TGCTGGCATTCT CTTCTG(SEQ ID NO: 18) HMG- 09-0-2807 AACGACGCGAA AF127919 (f: 176-KBrB123C07R, n/a OEG82B0 I/Y CGGTTGCAACAA 201; r: 678-649; KBrB078D23F,5.B1, GAC 503 bp) KBrB026F21R, BOMRW (SEQ ID NO: 38) CT012477 66TR,09-0-2808 CGTCAACTTTAG BONPC3 CAACCAACAGG 2TF CACCATC (SEQ ID NO: 39)

TABLE 4 Real-time PCR Primers and Probes. Asterisk notes that all probes were labeled as described in the reference, except the HMG-I/Y probe; VIC-TAMRA was used instead ofHEX-TAMRA. Double asterisk notes that FatA(A) describes the A-genome specific assay disclosed herein. Final conc. Primer/(nM) in real- Gene Probe* Sequence (5′ to 3′) time PCR Reference CruAMDB510 GGCCAGGGTTTCCGTGAT 200 1 (SEQ ID NO 40) MDB511CCGTCGTTGTAGAACCATTGG 200 (SEQ ID NO 41) TM003 VIC- 200AGTCCTTATGTGCTCCACTTTCTGGTGC A-TAMRA (SEQ IN NO 42) FatA FatA-FGGTCTCTCAGCAAGTGGGTGAT 150 2 (SEQ ID NO 43) FatA-RTCGTCCCGAACTTCATCTGTAA 150 (SEQ ID NO 44) FatA-P FAM-  50ATGAACCAAGACACAAGGCGGCTTCA- TAMRA (SEQ ID NO 45) FatA(A)** 09-0-3249ACAGATGAAGTTCGGGACGAGTAC 300 Described  (SEQ ID NO 19) in this 09-0-3251CAGGTTGAGATCCACATGCTTAAATAT 900 patent  (SEQ ID NO 20) application.09-QP87 FAM-AAGAAGAATCATCATGCTTC-MGB 150 (SEQ ID NO 21) HMG-I/Y hmg-FGGTCGTCCTCCTAAGGCGAAAG 500 3 (SEQ ID NO 46) hmg-R CTTCTTCGGCGGTCGTCCAC500 (SEQ ID NO 47) hmg-P** VIC-CGGAGCCACTCGGTG 300 CCGCAACTT-TAMRA (SEQ ID NO 48) BnACCg8 acc1 GGTGAGCTGTATAATCGAGCGA 300 4(SEQ ID NO 49) acc2 GGCGCAGCATCGGCT 300 (SEQ ID NO 50) accp VIC- 200AACACCTATTAGACATTCGTTCCATTGG TCGA-TAMRA (SEQ ID NO 51) PEP pep-FCAGTTCTTGGAGCCGCTTGAG 300 5 (SEQ ID NO 52) pep-R TGACGGATGTCGAGCTTCACA300 (SEQ ID NO 53) pep-P FAM- 200 ACAGACCTACAGCCGATGGAAGCCTGC-TAMRA (SEQ ID NO 54)

REFERENCES IN TABLE 4

-   1. EURL method for detection of: 1) T45    (http://gmo-crl.jrc.ec.europa.eu/summaries/T45_validated_RTPCR_method.pdf), 2)    MS8    (http://gmo-crl.jrc.ec.europa.eu/summaries/Ms8_validated_Method_Corrected%20version%201.pdf), 3)    RF3    (http://gmo-crl.jrc.ec.europa.eu/summaries/Rf3_validated_Method.pdf),    and 4) RT73    (http://gmo-crl.jrc.ec.europa.eu/summaries/RT73_validated_Method.pdf)-   2. Wu, Y., Wu, G., Xiao, L., Lu, C. Event-Specific Qualitative and    Quantitative PCR Detection Methods for Transgenic Rapeseed Hybrids    MS1×RF1 and MS1×RF2; J. Agric. Food Chem. 2007, 55, 8380-8389-   3. Weng, H.; Yang, L.; Liu, Z.; Ding, J.; Pan, A.; Zhang, D. Novel    reference gene, High-mobility-group protein I/Y, used in qualitative    and real-time quantitative polymerase chain reaction detection of    transgenic rapeseed cultivarsl J. AOAC Int. 2005, 88, 577-584-   4. Hernandez, M.; Rio, A.; Esteve, T.; Prat, S.; Pla, M. A    rapeseed-specific gene, Acetyl-CoA Carboxylase, can be used as a    reference for qualitative and real-time quantitative PCR detection    of transgenes from mixed food samples. J. Agric. Food Chem. 2001,    49, 3622-3627.-   5. Zeitler, R.; Rietsch, K.; Vaiblinger, H. Validation of real-time    PCR methods for the quantification of transgenic contaminations in    rapeseed; Eur Food Res Technol (2002)214:346-351.

CruA, HMG I/Y and FatA PCR Design and Selection of Template DNAs

In order to design an A-genome specific endogenous real-time PCR assay,three genes were selected: CruA, HMG-I/Y and FatA. Amplification andsequencing of an approximately 500 bp region from these genes frommultiple varieties of the six species in the Triangle of “U” wasperformed. Primers were designed in the identified conserved regions inthe available sequences from the six members of the Triangle of “U”.Although sequences were not available from all six of the Brassicaspecies for all three genes, alignments were made and primers weredesigned in the conserved regions of the available sequences. Seeds fromseveral varieties (N=38) were selected from various geographical regionsin order to capture sequence diversity of the A-genome species (B. rapa,B. napus, and B. juncea) to develop the assays. The number of varietiessequenced from the remaining 3 species (B. olearcea, B. carinata, and B.nigra) were less (N=15).

Primer Design for PCR/Cloning/Sequencing Regions of FatA, CruA and HMGGenes

GenBank sequences of FatA, CruA and HMG from the Triangle of “U” specieswere selected and aligned to find conserved sequences for designingprimers and to amplify an approximate 500 bp region from each of thesegenes. Table 3 shows the Genbank accession number of the sequences usedin the alignment, as well as the position and amplicon size of theselected primers (on the B. napus accession). No GenBank sequences wereavailable for B. carinata and B. nigra for these three genes.

Primers were selected to PCR, clone and sequence a region of each gene.For CruA, the selected primers (09-O-2809/09-O-2811) encompass thereferenced real-time assay (MDB410, MDB511, TM003), extending 599 bases5′ and 47 bases 3′ of the real-time PCR amplicon. For HMG-I/Y theselected primers (09-O-2807/09-O-2808) encompass the referencedreal-time assay (hmg-F, hmg-R, hmg-P), extending 273 bases 5′ and 131bases 3′ of the real-time PCR amplicon. For FatA, the selected primers(09-O-2812/09-O-2813) are slightly downstream of the FatA assay (FatA-F,FatA-R, FatA-P), omitting 32 bases 5′ and extending 462 bases 3′ of thereal-time PCR amplicon.

PCR, Cleanup and Cloning

Genomic DNA (100-120 ng) isolated from 53 seed varieties (see italicizedvarieties in Table 1) was used as template for PCRing the region ofinterest to be cloned and sequenced. The selection of seeds consist ofmultiple varieties from various geographical regions for B. napus(N=17), B. juncea (N=9), B. rapa (N=12), B. nigra (N=3), B. carinata(N=2) and B. oleracea (N=10). The purified PCR products were cloned intoPGEM-T Easy vector. Approximately 6 clones were selected for sequencingusing the T7 and SP6 vector primers. In some cases more clones wereisolated and sequenced to achieve coverage of both genomes in theallotetraploid species.

Sequence Analysis and Optimization of A-Specific Real-Time PCR Primersand Probe

Sequencher v. 4.8 was used for sequence analysis of the cloned PCRproducts from the CruA, HMG and FatA genes. Alignments of the finalcontigs were made in Vector NTI. After the design of severalprimer/probe combinations to be specific to the A-genome of FatA, theoptimum primer/probe combination was selected based on A-genomespecificity, cycle threshold (Ct) values and PCR efficiency. The optimumprimer and probe concentrations were selected based on Ct values, deltaRn values, and PCR efficiency.

Once the assay was optimized, a dilution series was prepared with B.napus genomic DNA. A 40 ng/ul dilution of genomic DNA was seriallydiluted 4 times at 1:2. This dilution series was tested in the optimizedreal-time assay, using 5 ul of the dilutions (input template DNA in thePCR ranged from 200 ng to 12.5 ng). PCR efficiency and R2 coefficientwere evaluated.

Specificity Testing with Six Endogenous Real-Time PCR Assays

Six rapeseed endogenous real-time PCR assays were selected for thespecificity testing, including the FatA (A) assay described herein. Forconsistency, all reactions included 15 ul of a mastermix (includingprimers, probes, water and 10 ul of Applied Biosystems TaqMan UniversalPCR Master Mix w/o AmpErase UNG) and 5 ul of 20 ng/ul genomic DNA (=100ng genomic DNA) for a final reaction volume of 20 ul. The primer andprobe concentrations were as described in the relevant references, andlisted in Table 4.

The cycling parameters for the real-time PCR runs were as follows:initial denaturation at 95° C. for 10 minutes; 40 cycles of 95° C. for15 seconds (denaturation) and 60° C. for 60 seconds (annealing andextension). The real-time PCR runs were performed on 384-well plates inan Applied Biosystems 7900HT instrument. Data was analyzed using AppliedBiosystems Sequence Detection Systems (SDS) v. 2.3. For most of thetested samples (see Table 1), the PCR was run in triplicate over threeseparate real-time PCR runs. Some of the DNA was in limited supply andwas run in duplicate and/or less PCR runs. See Table 2 for descriptionof the number of runs and replicates in each of the assays with the DNAthat was in limited supply. Varieties NW4219BC, 469735, JS0879BC,JS0917BC, and 597829 had less replicates due to poor PCR results, notlack of DNA.

Example 2 Sequencing Regions of CruA, HMG I/Y and FatA

The primers that were selected resulted in amplification in all sixspecies. To maximize the likelihood that amplification would occur onboth genomes within the allotetraploid species (B. napus, B. juncea andB. carinata), additional clones were selected.

Once the PCR products were cloned from the multiple varieties within thesix species, several clones (≧6) were selected for sequencing in orderto increase the chance of capturing all potential diversity from theresulting PCR products. For the allotetraploid species, the goal was toobtain sequences from both genomes.

Subsequent to sequence analysis, some additional clones were selectedand sequenced in order to fill gaps in genome coverage for theallotetraploid species.

Example 3 Assigning Sequence Data into Consensus Representing Genomes

The consensus sequences were assigned to various consensus groups, andbased on overlap of the base species (B. rapa, B. oleracea and B. nigra)and the allotetraploid species (B. napus, B. juncea and B. carinata),these were further assigned to either the A, B or C genome. Due togenetic variation of the A, B and C genomes between the species, in somecases more than one consensus sequence was defined for each genome.

CruA

For the CruA sequencing project, two A and two C genome consensus wereidentified. However, defining a consensus for the B genome wasdifficult. The cloned PCR products from B. juncea (AABB) all fell withinthe A consensus. In addition, several of the B. juncea varieties weredifficult to PCR. The two B. carinata (BBCC) varieties resulted incloned PCR products from the C genome only. The difficulty indetermining a B consensus may be due to poor binding of the primers tothe B genome or the B sequence may not differ significantly from the Aconsensus. The limited number of varieties from B. carinata (BBCC) andB. nigra (BB) that were sequenced also may have contributed todifficulty in distinguishing a B consensus. Sequences from ACNO 633153were assigned to both the A and C contigs, suggesting this variety wasmis-typed, and is actually a B. napus (AACC) variety. Two B. nigravarieties (ACNO 633142 and ACNO 649156) were assigned to 2 differentconsensus: 633142 to a non-distinguishing consensus and 649156 to the Aconsensus, suggesting that these two varieties are not the same species.Within the two A genome consensuses from CruA, there was not a suitableconserved region for the design of a real-time assay.

HMG

For the HMG sequencing project, four A genome consensus, and one Cgenome consensus were identified. Again, a B consensus could not beidentified, either because it could not be distinguished from the A andC consensus, or because it could not be amplified with the PCR primers.All sequences from the B. juncea varieties (AABB) were assigned to the Aconsensus, except ACNO 458942 which was assigned to the A and C. The B.nigra varieties (BB) were assigned to the A genome, and the B. carinatavarieties (BBCC) were assigned to the C consensus. Inability to get aconsensus for the B genome could be due to inefficient binding of theprimers to the target on the B genome, or overlap with the A consensus.A small sample size of the B. nigra and B. carinata varieties weresequenced which may have made it more difficult to come up with a Bconsensus. As for CruA, ACNO 633153 grouped with sequences assigned tothe A and C genome consensus. The two B. nigra species grouped into twodifferent consensuses: ACNO 633142 did not group in the A or C consensusand ACNO 649156 grouped into the A consensus. A conservedA-genome-specific region could not be identified from the sequencedregion of HMG.

FatA

For FatA, there were six consensus sequences: three for the A-genome,two for the C genome, and one for the B genome. The sequence from ACNO633153 grouped into the C1 and A1 contigs, verifying that it is a B.napus and not a B. rapa variety. In addition, ACNO 649156 sequencegrouped within the A and B consensus, verifying that it is a B. junceavariety and not a B. nigra. The six consensus sequences from FatA werealigned in VNTI, and the presumed evolutionary relationship of the sixsequences is displayed in FIG. 2-A. FIG. 2-B shows the distribution ofthe various species into the six consensuses.

Example 4 Design and Optimization of A-Specific Real-Time PCR Assay

Several real-time primers and probes were designed in regions conservedamong FatA of all three A consensus, and divergent from the FatA of theB and C genomes. The primer/probe set that gave the lowest Ct value andthe highest PCR efficiency was selected for further analysis. Theselected primers and probe are shown on the consensus alignment in FIG.3.

After a test for specificity on a smaller set, the real-time PCR assaywas optimized. Three runs were performed on a dilution series of DNAfrom B. napus variety 45H73. The results of these runs are shown inTable 5.

TABLE 5 Slope and R2 from 3 real time PCR runs with the FatA(A) assay.Run Slope R2 PCR Efficiency (%) 1 −3.39 0.997 97.2 2 −3.42 0.997 96.1 3−3.41 0.996 96.5

Example 5 Specificity Comparison of Six Rapeseed Endogenous Real-TimePCR Assays

To verify that the FatA(A) real-time PCR assay offers greaterspecificity than existing systems, a panel of DNAs were tested with theFat(A) assay as well as five other rapeseed endogenous real-time PCRassays. The sources of DNA included: 1) the seed varieties used in thesequence analysis (ACNO 649156 was omitted); 2) additional varieties ofB. carinata and B. nigra; 3) other species that may contaminate canolafields; and 4) seeds from various other crops. The real-time assays wererun using the primers and probes (and final concentrations in thereactions) as described in references for the various assays describedin Table 4. In most cases the real-time assay was run in triplicate over3 separate PCR runs. Due to variation in DNA yields, some DNAs were inlimited supply and had fewer replicates and/or runs (see Table 2). Theaverage Ct values from this specificity testing is shown in Table 6.

With one exception (B. nigra variety ACNO 273638), the FatA(A) assayresulted in average Ct values of ≧35 for all DNAs tested except theBrassica species containing the A-genome: B. napus, B. rapa and B.juncea varieties. The average Ct value of the ACNO 273638 B. nigravariety in the FatA(A) assay is approximately 9 cycles later than theother A-genome samples, therefore it was possible that this B. nigrasample was contaminated with one of the A-genome species. The A genomewas not detected during the sequencing of this variety, but if theamplification is due to contaminating A genomic DNA, it is at a very lowlevel based on the delayed Ct value.

All of the other assays tested showed less specificity than the FatA(A)assay; for example, cross reactivity with non-Brassica species and/ornot specific to the A-genome. BnACCg8 detects the A and B genome andSinapis alba, Sinapis arvenis (and Sinapis arvensis ssp. arvensis); PEPdetects the A and C genome; and, and HMG detects primarily the A and Bwith some cross-reactivity with the C genome and cross reactivity withsome of the Sinapis arvensis varieties. The CruA assay appears to detectall A, B, and C genomes as well as the related species Thlaspi arvense,Erucastrum gallicum, Raphanus raphanistrum, Raphanus sativus, Sinapisalba, and Sinapis arvensis (and the subspecies: Sinapis arvensis ssp.arvensis). The FatA assay detects the A, B and C genomes, as well asCamelina sativa, Raphanus raphanistrum, Raphanus sativus, Sinapis alba,Sinapis arvensis (and the subspecies: Sinapis arvensis ssp. arvensis)and Arabidopsis thaliana.

TABLE 6 Average Ct values with six endogenous real-time PCR systems.ACNO or Average Ct values Species Variety FatA(A) BnACCg8 CruA FatA HMGPEP B. napus 45H73 21.2 21.9 21.9 20.3 21.5 20.1 NS1822BC 21.8 22.9 22.620.9 22.2 20.6 46A76 21.6 22.0 22.3 20.6 22.0 20.4 NS5536BC 22.2 22.723.1 21.2 22.6 21.1 43A56 22.5 23.1 23.4 21.5 23.9 21.8 NW1717M 21.722.8 22.5 20.6 27.5 21.2 NW4219BC 22.1 22.6 23.0 20.5 23.6 21.5 NW4201BC21.6 22.1 22.4 20.7 22.2 21.0 436554 21.9 22.6 22.9 21.0 25.5 21.3458967 21.9 22.4 22.9 20.9 26.0 21.3 458954 22.2 22.9 23.3 21.2 23.321.6 531273 21.9 22.9 23.3 20.8 23.4 21.5 458941 21.8 23.1 22.7 20.823.3 21.2 469735 21.7 22.3 22.6 20.2 25.2 21.1 458605 21.8 23.0 22.720.9 23.4 21.0 311729 22.4 22.5 23.0 21.3 22.8 21.5 305278 21.7 22.322.7 20.8 27.5 20.8 B. rapa Tobin 22.1 22.6 22.9 21.1 22.8 21.7 Klondike21.0 21.3 23.0 21.8 22.4 21.1 Reward 20.7 21.0 22.6 21.4 21.9 20.8 41P9521.1 21.2 23.1 21.7 22.1 21.2 257229 21.6 21.7 23.6 22.5 22.7 21.8633153 21.1 22.5 23.2 22.2 23.1 21.5 649159 21.5 21.9 23.4 22.3 22.222.1 163496 21.2 21.7 23.4 22.0 22.4 22.1 B. rapa ssp 347600 21.5 22.723.4 22.1 22.6 21.0 dichotoma B. rapa ssp 649190 21.7 21.9 23.5 22.222.4 22.4 oleifera B. rapa ssp 346882 21.1 23.0 23.3 21.7 22.1 20.9trilocularis B. rapa var. 390962 21.2 23.1 23.2 22.0 21.9 22.2parachinensis B. juncea JS0879BC 22.0 24.1 23.6 20.7 23.5 23.4 JS0917BC22.0 23.8 23.4 20.7 23.3 23.5 JS0936BC 21.9 24.0 23.9 21.4 23.7 23.4JS1056BC 21.5 23.6 23.2 20.9 23.0 22.7 JS1260MC 21.6 23.6 23.2 20.9 23.222.8 JS1432MC 21.7 23.5 23.2 21.0 23.2 22.9 418956 21.7 23.7 23.5 20.823.4 23.1 458942 22.0 23.3 23.6 21.1 23.9 23.2 603011 21.7 23.8 23.520.9 22.9 23.2 B. oleracea 249556 UDT UDT 23.1 20.4 33.2 21.7 var.alboglabra B. oleracea 28888 UDT 37.5 22.8 21.1 32.7 21.5 var. botrytisB. oleracea 29041 UDT 37.5 23.3 21.1 33.2 22.2 var. capitata B. oleracea29800 UDT 37.6 22.3 20.3 32.2 20.8 var. costata B. oleracea 365148 UDT37.5 22.5 20.6 31.9 21.1 var. gemmifera B. oleracea 29790 UDT 37.1 23.221.3 32.7 22.1 var. gongylodes B. oleracea 28852 UDT UDT 27.4 25.8 35.026.4 var. italica B. oleracea 30862 UDT 38.0 22.4 20.7 31.6 21.4 var.medullosa B. oleracea 30724 UDT 37.8 22.4 20.8 31.0 21.2 var. ramosa B.oleracea 32550 UDT 38.7 22.8 20.7 30.8 21.8 var. viridis B. nigra 27363830.1 25.1 24.9 19.6 25.3 UDT 633142 37.0 25.4 23.6 20.0 25.1 UDT 193960UDT 30.1 26.4 23.6 31.6 21.4 271444 UDT 27.7 25.0 21.0 28.0 UDT 633143UDT 25.8 23.5 20.4 27.1 UDT 220282 UDT 29.9 27.8 21.6 29.8 UDT 649154UDT 24.8 22.4 19.5 27.3 UDT 280638 UDT 26.8 26.5 20.5 27.1 UDT 35736937.4 26.7 25.3 20.5 26.9 UDT 633147 36.2 25.6 23.7 20.4 26.9 UDT 13151237.1 26.7 25.1 20.4 27.0 UDT 597829 UDT 25.6 23.7 20.3 26.2 UDT 649155UDT 27.3 24.2 20.5 27.8 UDT B. carinata 613124 UDT 26.7 23.1 20.7 27.822.3 597822 UDT 26.4 23.2 20.1 27.3 21.7 360879 UDT 25.6 25.6 19.9 26.5UDT 596534 UDT 26.6 22.6 20.0 27.6 21.5 2779 UDT 27.0 23.7 20.7 27.922.2 193460 UDT 26.7 23.4 20.8 28.5 23.0 633076 UDT 27.3 23.5 20.8 28.022.3 390133 UDT 26.9 22.8 20.5 27.2 22.2 209023 UDT 26.9 23.4 20.4 27.721.9 360882 UDT 27.1 23.5 21.0 26.8 22.5 Sinapis alba 19266 UDT 27.024.1 21.7 UDT UDT 305276 UDT 29.2 23.6 22.8 UDT UDT 311724 UDT 27.9 23.721.7 UDT UDT 458960 UDT 27.5 23.5 22.0 UDT UDT 409025 UDT 29.0 24.5 21.9UDT UDT 633274 UDT 25.2 23.5 20.8 UDT UDT Sinapis 21449 UDT 26.6 23.124.0 UDT UDT arvensis 597863 UDT 27.5 23.6 23.7 UDT 30.5 633374 UDT 27.124.4 24.6 29.5 UDT Sinapis 296079 UDT 26.5 23.6 23.9 29.5 UDT arvensis407561 UDT 26.0 22.6 23.3 29.8 UDT subsp. 633411 UDT 28.0 24.4 24.9 29.5UDT arvensis Erucastrum 22990 37.1 UDT 26.1 21.0 UDT UDT gallicumRaphanus 271456 UDT 37.0 27.8 24.1 UDT UDT raphanistrum Raphanus 268370UDT 35.6 27.9 24.0 UDT UDT sativus 650165 UDT UDT UDT 22.8 39.1 UDTThlaspi 633414 UDT UDT 32.1 UDT UDT UDT arvense 29118 UDT UDT 31.7 UDTUDT UDT Arabidopsis UDT UDT UDT 29.8 UDT 37.7 thaliana Columbia typeGossypium UDT UDT UDT 37.3 UDT UDT hirsutum Helianthus 7451 39.0 UDT UDTUDT UDT UDT annuus 29348 UDT UDT UDT UDT UDT UDT 592319 UDT UDT UDT UDTUDT UDT Solanum 645248 UDT UDT UDT UDT UDT UDT lycopersicum Glycine maxUDT UDT UDT UDT UDT UDT Oryza sativa UDT UDT UDT UDT UDT UDT Sorghumbicolor UDT UDT UDT UDT UDT UDT Zea mays UDT UDT UDT UDT UDT UDT UDTrepresents undetermined, or not detectable in this assay.

Example 6 Testing for Heterogeneity within Each A-Genome Species

In addition to specificity, a desirable endogenous reference system maynot exhibit allelic variation among varieties. The FatA(A) assayrecognizes the A-genome, and therefore three different species, withvarying haploid genome sizes. Therefore, the stability measurements wererestricted to the comparison of Ct values of the varieties within eachspecies. By restricting the Ct comparison to those varieties within aspecies, it eliminates the Ct variation caused by the variation ingenome size between the base species (B. rapa, AA) and theallotetraplied species (B. juncea/AABB and B. napus/AACC). The resultsof this analysis are shown in Table 7. For B. napus, the Ct valuesranged from 21.6 to 22.5; for B. rapa the Ct values ranged from 20.7 to22.1, and for B. juncea the Ct values ranged from 21.5 to 22.0. In theB. napus and B. juncea species, the Ct range is within 1 Ct value; andfor B. raja the Ct range is within 1.4 Cts. In all cases the deviationfrom the mean is within 1 Ct value.

TABLE 7 Heterogeneity Test of FatA (A-genome specific) endogenousreference real-time PCR assay. ACNO or Mean Variety (within Species nameMean species) SD CV (%) B. napus 45H73 21.2 21.84 0.36 1.66 NS1822BC21.8 46A76 21.6 NS5536BC 22.2 43A56 22.5 NW1717M 21.7 NW4219BC 22.1NW4201BC 21.6 436554 21.9 458967 21.9 458954 22.2 531273 21.9 45894121.8 469735 21.7 458605 21.8 311729 22.4 305278 21.7 633153** 21.1 B.rapa Tobin 22.1 21.34 0.39 1.82 Klondike 21 Reward 20.7 41P95 21.1257229 21.6 649159 21.5 163496 21.2 B. rapa ssp 347600 21.5 dichotoma B.rapa ssp oleifera 649190 21.7 B. rapa ssp 346882 21.1 trilocularis B.rapa var. 390962 21.2 parachinensis B. juncea JS0879BC 22 21.79 0.190.88 JS0917BC 22 JS0936BC 21.9 JS1056BC 21.5 JS1260MC 21.6 JS1432MC 21.7418956 21.7 458942 22 603011 21.7 Note: Sequences from ACNO 633153 wereassigned to both the A and C contigs, suggesting this variety mayactually be a B. napus (AACC) variety (see Example 3 herein).

While the foregoing has been described in some detail for purposes ofclarity and understanding, it will be clear to one skilled in the artfrom a reading of this disclosure that various changes in form anddetail can be made without departing from the true scope. For example,all the techniques, methods, compositions, apparatus and systemsdescribed above may be used in various combinations. All publications,patents, patent applications, or other documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, or other document were individually indicated to beincorporated by reference for all purposes.

What is claimed is:
 1. A method of detecting and quantifying the amountof Brassica A genomic DNA in a sample, the method comprising: (a)specifically amplifying a genomic DNA fragment of the Brassica A genome,wherein the amplified DNA fragment comprises at least one of thenucleotide sequences of a genomic region selected from the groupconsisting of SEQ ID NOS: 25, 26, 27, 28, and 35; and (b) detecting andquantifying the Brassica A genome from the amplified fragment of theBrassica A genome.
 2. The method of claim 1, wherein the amplifiedfragment of Brassica A genome comprises (i) the nucleotide sequence ofat least one of SEQ ID NOS: 29, 30, or 31; or (ii) a nucleic acidfragment of at least one of SEQ ID NOS: 29, 30, or 31, wherein thenucleic acid fragment is selected from the group consisting ofnucleotide position from about 50 to about nucleotide position 400, 50to about nucleotide position 100, 50 to about nucleotide position 350,400 to about nucleotide position 350, 400 to about nucleotide position200, and 400 to about nucleotide position 100 of SEQ ID NOS: 29, 30, or31.
 3. The method of claim 1, wherein the amplification is performedwith a primer pair comprising nucleotide sequences selected from thegroup consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and
 24. 4.The method of claim 1, wherein the genomic DNA comprises a FatA gene. 5.The method of claim 1, wherein the amplification does not substantiallyamplify Brassica B and C genomic DNA.
 6. A method of determining therelative amount of a Brassica transgenic event in a sample, the methodcomprising: (a) performing a Brassica A genome specific polymerase chainreaction, wherein the analysis includes specifically amplifying agenomic DNA fragment of the Brassica A genome, wherein the amplified DNAfragment comprises at least one of the nucleotide sequences of a genomicregion selected from the group consisting of SEQ ID NOS: 25, 26, 27, 28,and 35; (b) determining the total amount of Brassica A genomic DNA inthe sample; (c) performing an event specific assay for the transgenicevent to determine the amount of the transgenic event in the sample; and(d) comparing the amount of the transgenic event DNA to the total amountof Brassica A genomic DNA in the sample.
 7. The method of claim 1wherein the amplified fragment of Brassica A genome comprises thenucleotide sequence of at least one of SEQ ID NOS. 29, 30, or 31 fromnucleotide position about 50 to about
 400. 8. The method of claim 6,wherein the amplification is performed with a primer pair comprisingnucleotide sequences selected from the group consisting of SEQ ID NOS:17, 18, 19, 20, 21, 22, 23, and
 24. 9. The method of claim 6, whereinthe genomic DNA comprises a FatA gene.
 10. The method of claim 6,wherein the amplification does not substantially amplify Brassica B andC genomic DNA.
 11. A method of determining adventitious presence of aBrassica transgenic event in a sample, the method comprising: (a)obtaining a sample suspected of containing a Brassica transgenic event;(b) performing a Brassica A genome specific polymerase chain reaction,with a primer, wherein the primer binds to a genomic region of theBrassica A genome, the genomic region selected from the nucleotidesequence of at least one of SEQ ID NOS: 29, 30, or 31 from nucleotideposition about 50 to about 400; (c) determining the total amount ofBrassica A genomic DNA in the sample; (d) performing an event specificquantitative assay for the transgenic event to determine the amount ofthe transgenic event DNA in the sample; and (e) comparing the amount ofthe transgenic event to the total amount of Brassica in the sample. 12.The method of claim 11 wherein the primer is selected from the groupconsisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and
 24. 13. Anamplicon comprising at least one of the nucleotide sequences of agenomic region selected from the group consisting of SEQ ID NOS: 25, 26,27, 28, and 35 wherein the amplicon is not larger than 500 base pairs.14. An oligonucleotide comprising a nucleotide sequence selected fromthe group consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and 24wherein the oligo is about 15-500 nucleotides.
 15. A detection kitcomprising oligonucleotides comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 17, 18, 19, 20, 21, 22, 23, and24 wherein the oligo is less than about 50 nucleotides and one or morereaction components to perform a quantitative reaction.
 16. A method ofdetermining trait purity of a Brassica trait, the method comprising: (a)obtaining a sample of a Brassica trait; and (b) performing the BrassicaA genome specific assay of claim
 1. 17. The method of any one of claims6 and 11, wherein the trait is selected from the group consisting ofRT73, RT200, MON88302, DP-073496, HCN92, T45 (HCN28), 23-18-17, 23-198,OXY-235, MS1, MS3, MS6, MS8, RF1, RF2, RF3, and Topas 19/2.
 18. Themethod of any one of claims 1-17, wherein the determination comprisesperforming a quantitative polymerase chain reaction.
 19. A method ofestablishing purity of a Brassica seed lot, the method comprisingperforming a polymerase chain reaction wherein the oligonucleotideprimers or probes are capable of discriminating the Brassica A genomefrom the Brassica B and C genomes, wherein the oligonucleotide primersand/or probes bind to a target region of the Brassica A genome, thetarget region comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 25, 26, 27, 28, and
 35. 20. The method ofclaim 19, wherein the oligonucleotide primers and/or probes comprise anucleotide sequence selected from the group consisting of SEQ ID NOS:19, 20, 21, 22, 23, and
 24. 21. A method of quantifying the amount of atransgenic element in a Brassica sample, the method comprising: (a)performing a polymerase chain reaction wherein the oligonucleotideprimers or probes are capable of discriminating the Brassica A genomefrom the Brassica B and C genomes, the oligonucleotide primers or probesbind to a target region of the Brassica A genome comprising a nucleotidesequence selected from the group consisting of SEQ ID NOS: 29, 30, and31; (b) performing the transgenic element specific quantitativepolymerase chain reaction; and (c) determining the amount of thetransgenic element present in the canola sample by comparing to theamount of Brassica A genomic DNA in the sample.
 22. A method ofdetermining seed purity of a seed sample suspected of containing theBrassica A genome, the method comprising: (a) specifically amplifying agenomic DNA of the Brassica A genome, wherein the amplified DNAcomprises at least one of the nucleotide sequences of a genomic regionselected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and35; and (b) determining the purity of the seed sample based on thepresence or absence of the Brassica A genome
 23. The method of claim 22,wherein the seed sample contains at least one of broccoli, brusselsprouts, mustard seeds, cauliflower, collards, cabbage, kale, kohlrabi,leaf mustard, or rutabaga.
 24. A method of determining the presenceand/or quantity of the Brassica A genome, the method comprising: (a)specifically hybridizing DNA of the Brassica A genome with a probe,wherein the probe selectively binds to at least one of the nucleotidesequences of a genomic region selected from the group consisting of SEQID NOS: 25, 26, 27, 28, and 35, optionally under high stringencyconditions, and; (b) detecting the presence and/or quantity of theBrassica A genome.
 25. The method of claim 24, wherein the probe bindsthe genomic region of the Brassica A genome selected from the nucleotidesequence of at least one of SEQ ID NOS: 29, 30, or 31 from nucleotideposition about 50 to about
 400. 26. The method of claim 24, wherein theprobe comprises the nucleotide sequences of SEQ ID NOS: 19, 20, 21, 22,23, or
 24. 27. A method of sequencing a region of the Brassica A genome,the method comprising: (a) obtaining a DNA sample; and (b) performing asequencing reaction of the DNA sample wherein the sequenced regioncomprises at least one of the nucleotide sequences of a genomic regionselected from the group consisting of SEQ ID NOS: 25, 26, 27, 28, and35.